The present invention relates to DNA sequences and plasmids, containing these DNA sequences, which by integration into a plant genome, cause the activity of the sucrose-phosphate-synthase (SPS) of the plant to be changed and thus affect the sugar metabolism of the plant. The invention further relates to transgenic plants, in which through introduction of the DNA sequences, changes in the activity of the sucrose-phosphate-synthase are produced.
Sucrose is of central importance for the plant and serves many functions. For the long distance transport of photoassimilates and/or energy between various organs in plants, sucrose is almost exclusively used. The sucrose which is transported in a specific heterotrophic organ determines the growth and the development of this organ. Thus it is known, e.g. from EP 442 592, that transgenic plants, in which the transport of the sucrose away from the exporting leaves is inhibited by expression of an apoplastic invertase, shows a strong reduction in the growth of e.g. roots or tubers in the case of potato plants. For tobacco plants, the principal importance of sucrose for the long distance transport of energy carriers within the plant is described in von Schaewen et al, 1990, EMBO J 9: 3033-3044.
While it has been clearly shown that a reduction of the amount of sucrose imported in the heterotrophic organs, such as tubers and seeds, leads to loss of yield, it is not known whether an increase in the amount of sucrose in the photosynthetically active parts of the plant, mainly the leaves, leads to a better supply of heterotrophic organs and thus to an increase in yield.
A second central role for sucrose and/or the hexoses, glucose and fructose which are derived from sucrose, is in the protection of plants against frost damage at low temperatures. Frost damage is one of the main limiting factors in agricultural productivity in the northern hemisphere. Temperatures below freezing lead to the formation of ice crystals. Since the growing ice crystals consist of pure water, water is abstracted from the cells as the temperature falls.
This dehydration has at least two potential damaging results:
a) all dissolved substances within a cell are strongly concentrated and the cell contracts following the loss of water. Highly concentrated salts and organic acids lead to membrane damage;
b) with rehydration from dew, the previously contacted cells reexpand. The cell membrane also expands again. The volume expansion puts a heavy mechanical load on the membrane.
It is thus clear that a freezing/dew cycle can lead to severe membrane damage of the cells and thus to damage to the plant.
It thus appears worth trying to hinder the freezing. One possible strategy is to increase the formation of osmotically active substances in the cytosol of plant cells. This should lead to a lowering of the freezing point. Osmotically active substances include sucrose and/or the two hexoses which are derived from sucrose.
The increased formation of sucrose and/or the two hexoses at low temperatures is desirable in the growing plant. Another situation can exist in the harvested parts of a plant, especially in storage. For example, in potato tubers that are stored at 4-8xc2x0 C., hexoses (glucose) accumulate. It would appear to be sensible, to see this as the answer to a lowering of the temperature (xe2x80x9ccold-sweeteningxe2x80x9d).
The accumulation of sucrose and glucose has in the case of potato tubers economically undesirable results. Increased amounts of reducing sugars, such as glucose, in potatoes which are fried when preparing crisps, chips and the like, leads to an undesirable browning due to the Maillard reaction. Such products with a dark brown color are not generally acceptable to the consumer. Further the cooking strength is strongly dependent on the content of starch and/or its breakdown products which are important in determining the quality characteristics of the potato.
In relation to the economic aspects, sucrose thus possesses three especially important functions:
1 as the transport form for the distant transport of photoassimilates,
2 as an osmotically active substance with the desirable activity of lowering the freezing point in intact, growing plants, and
3 in the undesirable formation of reducing sugars in stored harvested parts of a plant, e.g. the potato tubers, as a result of low temperatures.
The biosynthesis pathways for the formation of sucrose, either from the primary photosynthesis products (in the leaf) or by breakdown of starch (in the storage organs e.g. of potatoes), are known. An enzyme in sucrose metabolism is sucrose-phosphate-synthase (SPS). It forms sucrose-6-phosphate from UDP-glucose and fructose-6-phosphate, which in a second step is converted to sucrose.
The isolation of SPS from maize and the cloning of cDNA from mRNA from maize tissue is known (EP 466 995). In this application, processes for the purification of a protein such as by centrifuging or homogenates, differential precipitation and chromatography are described. A 300 times enrichment of SPS from plant tissue has been described by Salerno and Pontis (Planta 142: 41-48, 1978).
In view of the significance of SPS for carbohydrate metabolism, it is questionable whether plants can tolerate a reduction in SPS activity in all or in certain organs. It is especially not known whether it is possible to produce transgenic plants with a reduced SPS activity. Also the use of SPS for the modification of the functions of sucrose for lowering the freezing point in intact plants and for the formation of reducing sugars in harvested parts is not known.
For the preparation of plants with reduced SPS activity, i.e. plants with changed sucrose concentration, it is necessary to make available an SPS coding region of such plant species, for which processes are described, whereby transgenic plants can be grown in large numbers. In as much as a reduction of SPS activity can be achieved, by selection from a large amount, the possibility exists of obtaining plants with such a phenotype. Further organ specific promoters for gene expression should exist for the plant species, by which the possibility of an organ specific reduction of the SPS activity could be investigated.
A species which fulfils the stated requirements is Solanum tuberosum. The genetic modification of Solanum tuberosum by gene transfer using Agrobacteria is well described (Fraley et al., 1985, Crit Rev Plant Sci 4: 1-46). Promoters for leaf specific (Stockhaus et al., 1989, Plant Cell 1: 805-813), tuber specific (EP 375 092) and wound inducing (EP 375 091) gene expression are known.
The present invention now provides DNA sequences with which changes of SPS activity are actually and demonstrably possible and with which the sucrose concentration in the plant can be modified. It is concerned with sequences which include the coding region of sucrose-phosphate-synthase (SPS) from Solanum tuberosum. 
These DNA sequences can be introduced in plasmids and thereby combined with steering elements for expression in eukaryotic cells. Such steering elements are, on the one hand, transcription promoters and, on the other hand, transcription terminators.
Each plasmid comprises:
a) a suitable promoter that ensures that the coding sequence is read off at the suitable time point and/or in a specified development stage in the transgenic plants or in specified tissues of transgenic plants,
b) at least one coding sequence, that
i) is coupled to the promoter so that RNA can be translated into protein, whereby the protein demonstrates enzymatic activity, that leads to a modification of the sucrose concentration in the plant, or
ii) is coupled to the promoter so that the non-coding strand is read off, which leads to the formation of a so-called xe2x80x9canti-sensexe2x80x9d RNA, which suppresses the formation of the coding protein of an endogenous gene in the plant which is involved in the sucrose biosynthesis, and
c) a non-coding termination sequence that contains the signals for the termination and polyadenylation of the transcript.
The present invention further provides plasmids in which include DNA sequences which change the SPS activity in the plant.
The coding sequences named under b) include the SPS sequences with the following nucleotide sequences:
All sequences are cDNA sequences and stem from a cDNA library of leaf tissue. The expression gene is the same in various plant tissues. As a promoter, there can generally be used any promoter which is active in plants. The promoter should ensure that the foreign gene is expressed in the plant. The promoter can be chosen so that the expression occurs only in specified tissues, at a determined time point in the plant""s development or at a time point determined by outside influences. The promoter can be homologous or heterologous to the plant. Suitable promoters are e.g. the promoter of the 355 RNA of the cauliflower mosaic virus, the patatin promoter B33 (Rocha-Sosa et al. (1989) EMBO J 8: 23-29) or a promoter that ensures expression only in photosynthetically active tissues. Other promoters can be used which ensure expression only in specified organs, such as the root, tuber, seed, stem or specified cell types such as mesophyllic, epidermal or transport cells. For hindering cold sweetening, suitable promoters are those which ensure an activation of the transcription is stored in harvested parts of the plants. For this, there can be considered cold induced promoters or such promoters that become active during the transition of the tuber from the phase where it stores material to the phase where it gives up material.